There is a great need for low cost detection of bioparticles in various applications like disease diagnostics in animals and plants, bioterrorism, food inspection, and scientific research. Samples in such applications are found in a liquid medium. These particles can range from a few nanometers (nm) in size to a few micrometers (μm) in size. Viewing these particles with the naked eye is difficult. While μm size particles can be viewed with a microscope, nanometer (nm) scale particles require SEM, TEM, or AFM techniques. Techniques that involve the use of a microscope or SEM, TEM, AFM are not low cost in nature.
At a simple level, color change or light emission is usually an indicator of positive detection. Most low cost tests involve combining a few drops of a reagent with a sample on filter paper that has a color standard. If the target particle is detected with significant quantity, the reagent renders the color to a desired shade on the scale. While it would be nice to have all color change indicating reagents for detection, it is not always possible. In some cases the result of detection may involve multiple steps. For example, the target displays an antigen and a fluorescent-tagged antibody is then used to selectively bind to the target molecule. Following such a binding, excitation has to be provided to the combination for the fluorescence to indicate the presence of a valid target particle.
Another problem arises when a nanoliter drop sample has only a few of the target particles. In such cases, using color change as an indicator is impractical. Therefore a fluoro-tag may be used. However, having only a few molecules limits the number of photons emitted from the sample drop. Methods to solve this problem include: 1) increasing the light intensity of each fluoro-tag; 2) increasing the number of target molecules (by using a larger sample size and concentrating the sample drop); 3) using a higher sensitivity photodetector and 4) using other techniques like electrical conductivity change detection. While option 4 is viable only in a few cases, option 1 has been implemented in recent products by using nanoparticles that usually offer higher luminosity. In low cost diagnostic products, using photomultipliers to increase the photon counting efficiency or using complex optics and manual positioning are ruled out due to their increased cost.
Fluorescence Detection
Fluorescence microscopes are available from vendors like Perkin Elmer, Hitachi, Spec, etc. Smaller handheld fluorometers are available from Turner biosystems (16). Bench top models that use a bank of emission and excitation filters and multiple excitation sources to scan the spectrum from 200 nm to 1200 nm and provide a plot of the excitation and emission spectra are also available. The microscopes use a photomultiplier tube and focusing optics to increase the sensitivity and provide a field of view. All these instruments operate on the sample and reagent being present in a cuvette. None of them use dielectrophoresis (DEP) for manipulating the particles. Most of the fluorescent tags are latex beads (15) that have a fluorophore attached. These particles are relatively large in size (μm).